Dodecanoic acid, 2,2-dimethyl-3-oxopropyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item 2,2-Dimethyl-3-Iauroyloxy-propanal was tested for genetic toxicity with three in-vitro tests: A bacterial mutation assay (Ames-test), a chromosome aberration assay with CH V79 cells, and a cell gene mutation test with mouse lymphoma cells. 2,2-Dimethyl-3-Iauroyloxy-propanal showed no mutagenic activity in any of these tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

February 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

19 May 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No target gene.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/D3 in stock cultures: 12 hrs, determined on January 13, 2003) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

without S9-mix:
4 hrs exposure period: 6.3, 12.5, 25.0, 50.0, 100.0 and 200.0 µg/mL
18 hrs exposure period: 1.6, 3.1, 6.3, 12.5, 25.0 and 50.0 µg/mL
28 hrs exposure period: 6.3, 12.5, 25.0 and 50.0 µg/mL

with S9-mix:
4 hrs (I) exposure period: 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL
4 hrs (II) exposure period: 6.3, 12.5, 25.0, 50.0, 100.0 and 200.0 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; with and without metabolic activation (rat liver S9-mix)

DURATION
- Exposure duration: 4, 18, 28 hrs
- Expression time (cells in growth medium): 5 x 10E05 cells per flask

SELECTION AGENT (mutation assays): no

NUMBER OF REPLICATIONS: two per dose

NUMBER OF CELLS EVALUATED: 100 metaphase plates

DETERMINATION OF CYTOTOXICITY
- Method: precipitation:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher´s exact test (9) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications.The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

NA

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/mL were applied. In the absence of S9 mix, clear toxic effects were observed after 4 hrs treatment with 46.9 µg/mL (44 % of control) and above and after 24 hrs continuous treatment with 23.4 µg/mL (26 % of control) and above. In contrary, in the presence of S9 mix no clear toxic effects were observed after 4 hrs treatment up to the highest applied test item concentration .

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 187.5 µg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 350 mOsm, pH 7.4 versus 357 mOsm and pH 7.2 at 3000 µg/mL).

In experiment I, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 400 µg/mL. In experiment II at preparation interval 28 hrs precipitation of the test item in culture medium was observed after 28 hrs continuous treatment with 25 µg/mL and above in the absence of S9 mix and after 4 hrs treatment with 200 µg/mL in the presence of S9 mix.

In this study, in the absence of S9 mix toxic effects indicated by clearly reduced cell numbers or mitotic indices were observed in all experimental parts. In detail, strongly reduced cell numbers were observed in experiment II after 18 hrs continuous treatment with 25 µg/mL (48 % of control) . In all additional experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage .      In contrary, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied test item concentration being in the range of test item precipitation . In both cytogenetic experiments, in the absence and the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed . The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).                                  

In both experiments,EMS (200 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, being in the range of the historical control data.

In conclusion, it can be stated that under the experimental conditions reported, the test item 2,2-Dimethyl-3-lauroyloxy-propanal did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations (without metabolic activation) and to clearly precipitating concentrations (with metabolic activation).

Conclusions:

The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, 2,2-Dimethyl-3-lauroyloxy-propanal is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations (without metabolic activation) or to precipitating concentrations (with metabolic activation).

Executive summary:

The test item 2,2-Dimethyl-3-lauroyloxy-propanal, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitroin the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in experiment I, without metabolic activation, where only 50 metaphase plates were scored and for the test item concentrations 50 and 100 µg/mL in experiment II, with metabolic activation, where 200 metaphase plates were scored.

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/mL were applied. In the absence of S9 mix, clear toxic effects were observed after 4 hrs treatment with 46.9 µg/mL (44 % of control) and above and after 24 hrs continuous treatment with 23.4 µg/mL (26 % of control) and above. In contrary, in the presence of S9 mix no clear toxic effects were observed after 4 hrs treatment up to the highest applied test item concentration.

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 187.5 µg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 350 mOsm, pH 7.4 versus 357 mOsm and pH 7.2 at 3000 µg/mL).

In experiment I, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 400 µg/mL. In experiment II at preparation interval 28 hrs precipitation of the test item in culture medium was observed after 28 hrs continuous treatment with 25 µg/mL and above in the absence of S9 mix and after 4 hrs treatment with 200 µg/mL in the presence of S9 mix.

In this study, in the absence of S9 mix toxic effects indicated by clearly reduced cell numbers or mitotic indices were observed in all experimental parts. In detail, strongly reduced cell numbers were observed in experiment II after 18 hrs continuous treatment with 25 µg/mL (48 % of control). In all additional experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrary, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied test item concentration being in the range of test item precipitation.

In both cytogenetic experiments, in the absence and the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).                                  

In both experiments, EMS (200 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, being in the range of the historical control data.

In conclusion, it can be stated that under the experimental conditions reported, the test item 2,2-Dimethyl-3-lauroyloxy-propanal did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations (without metabolic activation) and to clearly precipitating concentrations (with metabolic activation).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-13 to 2011-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S9-mix

Test concentrations with justification for top dose:

3-hour treatment, in absence of exogenous metabolic activation (-S9):
15; 25; 30; 35; 40 and 50 μg/mL.
3-hour treatment, in presence of exogenous metabolic activation (+S9):
50; 80; 90; 100; 120 and 140 μg/mL.
The following concentrations were investigated in the Assay 2:
24-hour treatment (-S9 Mix): 5; 10; 12.5; 15; 20 and 25 μg/mL;
3-hour treatment (+S9 Mix): 50; 80; 90; 100; 110 and 115 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
The appropriate vehicle, Dimethyl sulfoxide (DMSO) was chosen based on the results of the preliminary solubility test. The test item solutions were prepared in DMSO and diluted prior to treatment. This vehicle was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 and 24 h
- Expression time: 2 days
- Selection time: 14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED:
-Expression Period
The cultures were maintained in flasks for 2 days, during this time the tk-/- mutation was expressed. During the expression period, subculturing was performed daily with the aim of not exceeding 1E06 cells per mL and retaining a total of at least 5E06 cells/flask. For this reason cell densities were adjusted to a concentration of 2x105/mL and transferred to flasks for further growth.
From observations on recovery and growth of the cultures during the expression period, at least five test dose levels plus negative and positive controls were plated for viability and 5-trifluorothymidine (TFT) resistance.

- Plating for Viability
At the end of the expression period, the cell density in the selected cultures were determined and adjusted to nominally 1x104/mL with RPMI 20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL as follows:
Initial Cell Concentration: 1E04/mL(A)
1. Dilution Step: 0.5 mL of Cell Suspension (A) + 9.5 mL of RPMI 10
Intermediate Cell Concentration: 5E02/mL (B)
2. Dilution Step: 0.8 mL of Cell Suspension (B) + 49.2 mL of RPMI 20
Final Cell Concentration: 8/mL
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microtiter plates (192 wells) resulting in an average of 1.6 cells per well. Microtiter plates were incubated at 37 ºC ± 1 °C containing approximately 5 % (v/v) CO2 in air for 12-14 days. Wells containing viable clones were identified by the unaided eye using background illumination and counted.

- Plating for 5-trifluorothymidine (TFT) Resistance
At the end of the expression period, the cell concentration was adjusted to 1E04/mL in each culture.
The TFT (300 μg/mL) was diluted 100-fold in the cell suspensions to give a final concentration of 3 μg/mL. (e.g.: 89.1 mL of cell suspension + 0.9 mL of TFT). Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microtiter plates (384 wells) resulting in average 2E03 cells per well. The microtiter plates was incubated at 37 ºC ± 1 °C containing approximately 5 % (v/v) CO2 in air for about two weeks (14 days) and wells containing clones were identified as above and counted.
In addition, scoring of small (less than a quarter of the diameter of the well) and large and colonies was performed.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore et al.):
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value. [e.g.: If the vehicle control MF is 50E-066, then one of the test cultures must have an MF of at least (50+126) x 10-6 = 176E-06 to meet the GEF criterion for a positive call.]

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: Due to the S9 presence slight opalescence was observed immediately after the treatment and during the treatment.

RANGE-FINDING/SCREENING STUDIES:
The treatment concentrations for the first main mutation assay (Assay 1) were determined in the preliminary Toxicity Test. After the Solubility Test the Toxicity Test was performed according to the followings:
In the first Toxicity Test a 3-hour treatment in the presence and absence of S9 Mix was performed with the concentration levels of: 2500, 1000, 500, 100, 50 and 10 μg/mL. Strong toxic effect of the test item was obtained down to the concentration of 100 μg/mL, in absence of S9 Mix, and down to 500 μg/mL, in presence of S9 Mix. In this test no plating experiment followed the treatment.
In the second Toxicity Test a 3-hour treatment in the presence and absence of S9 Mix and a 24-hour treatment in absence of S9 Mix was performed with the concentration levels of:
3-hour, -S9 Mix: 80, 60, 50, 20, and 10 μg/mL;
3-hour, +S9 Mix: 400, 250, 100, 80 and 50 μg/mL;
24-hour, -S9 Mix: 50, 10, 5, 1, 0.5, 0.1 μg/mL.
In the preliminary Toxicity Test, single cultures were used and positive controls were not included. The treated cells were washed following treatment and re-suspended in 10 mL tissue culture medium.
The cell concentrations were adjusted to 8 cells/mL and, for each dose, 0.2 mL was plated into each well of a 96-well microtiter plate. The microtiter plates were incubated at 37 ºC ± 1 °C in a humidified incubator gassed with 5 % (v/v) CO2 in air for 7-8 days. Wells containing viable clones were identified by the unaided eye and counted. During the treatments no precipitation of the test item (with or without S9 Mix, either) was observed in the culture medium by the unaided eye. The obtained survival data was used to choose the dose levels for the first main assay with the aim of covering a concentration range from the maximum possible cytotoxicity to a low effect level.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequency of the negative (vehicle) control cultures were within the expected normal range (50-170 mutants per 106 viable cells) in the performed experiments in line with the historical controls (Table 23).
The mutation frequency (MF) of the DMSO vehicle control in the Assay 2 at the 24-hour treatment (-S9 Mix) was 117 (Table 12). The actual, laboratory historical control data range for DMSO vehicle control is: 66-93 (Table 23). The obtained value was above the corresponding historical control data range, however within the assay acceptance range. The higher mutation frequencies in this case did not have any effect on the validity of the performed Assay 2.
2. The positive control chemicals (Cyclophosphamide in the presence and 4-Nitroquinoline-N-oxide in the absence of S9 Mix) induced a statistically significant increase in the mutant frequency (2 Sample t-Test, α=0.01) (Tables 11 and 12). The increase was within the range indicated in the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The concentrations to be applied in the Assay 1 were chosen according to the solubility and cytotoxicity results of the pre-experiments.
The following concentrations were investigated in the Assay 1:
3-hour treatment, in absence of exogenous metabolic activation (-S9):
15; 25; 30; 35; 40 and 50 μg/mL.
3-hour treatment, in presence of exogenous metabolic activation (+S9):
50; 80; 90; 100; 120 and 140 μg/mL.
Although the cytotoxicity results of the evaluated Assay 1 fulfilled the assay acceptance criterion, the chosen concentration range produced from little or no toxicity to 80-90 % toxicity in absence as well as in presence of exogenous metabolic activation (±S9), the concentration levels were modified for the Assay 2.
The following concentrations were investigated in the Assay 2:
24-hour treatment (-S9 Mix): 5; 10; 12.5; 15; 20 and 25 μg/mL;
3-hour treatment (+S9 Mix): 50; 80; 90; 100; 110 and 115 μg/mL.

Conclusions:

The test item 2,2-Dimethyl-3-Iauroyloxy-propanal (Aldehyde L) was tested for genetic toxicity in vitro in a cell gene mutation test with mouse lymphoma cells. 2,2-Dimethyl-3-Iauroyloxy-propanal (Aldehyde L) did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Executive summary:

The test item 2,2-Dimethyl-3-Iauroyloxy-propanal (Aldehyde L) was tested for genetic toxicity in vitro in a cell gene mutation test with mouse lymphoma cells.

In this mutation assay the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2E05/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the Assay 1, in the absence and also in presence of S9 Mix dose-related toxicity of the test item was observed based on both, on the harmonised relative survival (harmonised RS) and on the relative total growth (RTG) values. There were differences between the harmonised RS and the RTG values in absence and in presence of S9 Mix. In absence of exogenous metabolic activation the concentration level of 50 μg/mL; in presence of S9 Mix the concentration levels of 140 and 120 μg/mL were out of the analysable range, but the guideline criteria for the number of analysable concentration levels (at least four) as well as the required cytotoxicity degree (10-20% relative survival or relative total growth) at the maximum concentration (in this case maximum analyzable concentration) were fulfilled.

At the concentration level of 120 μg/mL (+S9 Mix) the obtained mutation frequency differed statistically significantly from that of the vehicle control (Dunnett’s Test,α= 0.05), but remained far below the GEF criterion for positive call and at this concentration the high level of toxicity (~97 %, based on the harmonised RS) was observed, therefore the observed significance was not taken into consideration as biological relevant.

The phases of the Assay 2 accomplished the necessary concentration ranges for a valid assay. There were at least four analyzable concentration levels, where the highest concentration level was based on the required level of the caused cytotoxicity (80-90 %). The concentration levels examined were slightly modified in presence of S9 Mix, because of the observed high degree of toxicity obtained in the Assay 1. Despite the investigated slightly lower concentration levels the highest concentration level 115 μg/mL dropped out of the analyzable concentration range, due to the narrow test item effect concentration range.

In the Assay 2 in presence and also in absence of exogenous metabolic activation there were one or two concentration levels where the obtained mutation frequencies statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). The changes of the mutation frequencies were not dose-related, not biologically relevant, and the GEF criterion for positive call was not attained in any case.

Under the conditions of this study, test item 2,2-Dimethyl-3-Iauroyloxy-propanal (Aldehyde L) did not induce gene mutations in absence and presence of metabolic activation in the cultured mammalian cells used.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-19 to 2004-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

08 June 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Vogel-Bonner Medium E
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background:
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. Historical control values for spontaneous revertants per plate are as follows: (-S9) Salmonella typhimurium TA 98: 15-60, TA 100: 75-200, TA 1537: 3-30, TA 1535: 3-28

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Vogel-Bonner Medium E
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background:
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. Historical control values for spontaneous revertants per plate are as follows: (-S9) Escherichia coli WP2 uvrA: 8-50.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/ß-naphthoflavone-induced rat liver S9-mix

Test concentrations with justification for top dose:

5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

S. typhimurium: TA 1537, Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium: TA 100; TA 1535, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

S. typhimurium: TA 98, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

S. typhimurium: TA 100; TA 98; TA 1535; TA 1537 and E. coli WP2 uvrA, with metabolic activation.

Details on test system and experimental conditions:

Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The bacterial strains were cultured in nutrient broth. The selective medium was a minimal medium with 2% glucose.
An appropriate number of tubes of molten top agar were 3 per control or concentration level and they were prepared and kept at 45 °C. An equivalent number of minimal plates were also properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C).

The content of the tubes:
top agar: 2000 µL
solvent or solution of test item or reference controls: 50 µL
over-night culture of test strain: 100 µL
phosphate buffer (pH: 7.4) or S9 mix: 500 µL

This mixture was mixed and poured on the surface of minimal plates. For activation part of this study instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of a non-activation and an activation test conditions each of them with negative and positive controls.

For the pre-incubation method before the overlaying the test item, the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes, provided the direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 ºC by using a shaker. After the incubation the content of the tubes were added to the molten top agar prior to pouring onto the surface of minimal agar plates. For activation part of this study instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of a non-activation and an activation test conditions each of them with negative and positive controls.

After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

The test is considered acceptable if for each strain:
– the bacteria demonstrate their typical responses to crystal violet and ampicillin
– the control plates without S9 mix are within the historical control data range
– corresponding background growth on both negative control and test plates occurs
– the positive controls show a distinct enhancement over the control plate

A test item is considered mutagenic if:
– a dose–related increase in the number of revertants occur and/or
– a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

A biologically relevant increase is described as follows:
– if in strain TA 100 the number of reversions is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
– if in strains TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

No data

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of this Ames-test, Aldehyde L was considered not genotoxic.

Executive summary:

In order to investigate the potential of 2,2-Dimethyl-3-lauroyloxy-propanal for its ability to induce gene mutations a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA.

The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in experiment I and II based on the results obtained in the pre-experiment on toxicity: 5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate

An additional confirmatory test was carried out, because of the noted toxicity in the experiment II. The examined bacterium strains were: Salmonella typhimurium TA100, TA1537 and TA1535. The following concentrations were tested: 50.00; 15.81; 5.00; 1.58; 0.5; 0.16 µg/plate. The experiment was performed in the absence of a post-mitochondrial supernatant (S9). In this confirmatory experiment the pre-incubation method was also used.

Inhibitory, toxic effect of the test item was observed in both experiments (I and II).

In experiment I the inhibitory effect of the test item was observed in case of Salmonella typhimurium TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in S. typhimurium TA 1535 in the concentration range of 5000.00-158.11 µg/plate, in S. typhimurium TA 1537 and Escherichia coli WP2 uvrA only at the highest concentration level of 5000.00 µg/plate. In S. typhimurium TA 100 the reduction of revertant colony numbers was equivocal (+S9), it was observed at the concentration levels of 5000.00 and 158.11 µg/plate. The revertant colony numbers were slightly reduced without metabolic activation in case of S. typhimurium TA 1537 in the concentration range of 5000.00-500.00 µg/plate.

In the experiment II using the pre-incubation method, the inhibitory effect manifested stronger. The pre-incubation method is more sensitive than the plate incorporation assay. The revertant colony numbers compared to the solvent control plates and the background lawn development were reduced in the investigated Salmonella typhimurium strains. The inhibition was stronger in the non-activation part (-S9) of the experiment in case of Salmonella typhimurium TA 100, TA 1535 and TA1537. In case of the tester strains Salmonella typhimurium TA100 and Salmonella typhimurium TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) while in case of TA1535 it was observable down to the concentraion preceding the lowest concentration (15.81 µg/plate). In case of Salmonella typhimurium TA 98 inhibition was observed in presence of S9 Mix. The revertant colony numbers were reduced in the concentration range of 5000.00-158.11 µg/plate, the background lawn development was reduced in the concentration range of 5000.00-1581.14 µg/plate.

In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. The examined strains Salmonella typhimurium TA 100 and TA 1537 were inhibited without metabolic activation at the concentration range of 50.00-1.58 µg/plate, the Salmonella typhimurium TA 1535 at the concentration range of 50.00-5.00 µg/plate. At the lower concentrations no cytotoxic effects were noted.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with 2,2-Dimethyl-3-lauroyloxy-propanal at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the solvent control values were observed in all experimental phases of the study. However, there was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in the performed experiment.

The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range.

The reference mutagens showed a distinct increase of induced revertant colonies.

After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate.

The reported data of this mutagenicity assay show that, under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, 2,2-Dimethyl-3-lauroyloxy-propanal is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames-test:

In order to investigate the potential of 2,2-Dimethyl-3-lauroyloxy-propanal for its ability to induce gene mutations a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA.

The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in experiment I and II based on the results obtained in the pre-experiment on toxicity: 5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate

An additional confirmatory test was carried out, because of the noted toxicity in the experiment II. The examined bacterium strains were: Salmonella typhimurium TA100, TA1537 and TA1535. The following concentrations were tested: 50.00; 15.81; 5.00; 1.58; 0.5; 0.16 µg/plate. The experiment was performed in the absence of a post-mitochondrial supernatant (S9). In this confirmatory experiment the pre-incubation method was also used.

Inhibitory, toxic effect of the test item was observed in both experiments (I and II).

In experiment I the inhibitory effect of the test item was observed in case of Salmonella typhimurium TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in S. typhimurium TA 1535 in the concentration range of 5000.00-158.11 µg/plate, in S. typhimurium TA 1537 and Escherichia coli WP2 uvrA only at the highest concentration level of 5000.00 µg/plate. In S. typhimurium TA 100 the reduction of revertant colony numbers was equivocal (+S9), it was observed at the concentration levels of 5000.00 and 158.11 µg/plate. The revertant colony numbers were slightly reduced without metabolic activation in case of S. typhimurium TA 1537 in the concentration range of 5000.00-500.00 µg/plate.

In the experiment II using the pre-incubation method, the inhibitory effect manifested stronger. The pre-incubation method is more sensitive than the plate incorporation assay. The revertant colony numbers compared to the solvent control plates and the background lawn development were reduced in the investigated Salmonella typhimurium strains. The inhibition was stronger in the non-activation part (-S9) of the experiment in case of Salmonella typhimurium TA 100, TA 1535 and TA1537. In case of the tester strains Salmonella typhimurium TA100 and Salmonella typhimurium TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) while in case of TA1535 it was observable down to the concentraion preceding the lowest concentration (15.81 µg/plate). In case of Salmonella typhimurium TA 98 inhibition was observed in presence of S9 Mix. The revertant colony numbers were reduced in the concentration range of 5000.00-158.11 µg/plate, the background lawn development was reduced in the concentration range of 5000.00-1581.14 µg/plate.

In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. The examined strains Salmonella typhimurium TA 100 and TA 1537 were inhibited without metabolic activation at the concentration range of 50.00-1.58 µg/plate, the Salmonella typhimurium TA 1535 at the concentration range of 50.00-5.00 µg/plate. At the lower concentrations no cytotoxic effects were noted.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with 2,2-Dimethyl-3-lauroyloxy-propanal at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the solvent control values were observed in all experimental phases of the study. However, there was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in the performed experiment.

The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range.

The reference mutagens showed a distinct increase of induced revertant colonies. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate.

The reported data of this mutagenicity assay show that, under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, 2,2-Dimethyl-3-lauroyloxy-propanal is considered to be non-mutagenic in this bacterial reverse mutation assay.

 

Chromosome Aberration Assay:

The test item 2,2-Dimethyl-3-lauroyloxy-propanal, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in experiment I, without metabolic activation, where only 50 metaphase plates were scored and for the test item concentrations 50 and 100 µg/mL in experiment II, with metabolic activation, where 200 metaphase plates were scored.

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/mL were applied. In the absence of S9 mix, clear toxic effects were observed after 4 hrs treatment with 46.9 µg/mL (44 % of control) and above and after 24 hrs continuous treatment with 23.4 µg/mL (26 % of control) and above. In contrary, in the presence of S9 mix no clear toxic effects were observed after 4 hrs treatment up to the highest applied test item concentration.

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 187.5 µg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 350 mOsm, pH 7.4 versus 357 mOsm and pH 7.2 at 3000 µg/mL).

In experiment I, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 400 µg/mL. In experiment II at preparation interval 28 hrs precipitation of the test item in culture medium was observed after 28 hrs continuous treatment with 25 µg/mL and above in the absence of S9 mix and after 4 hrs treatment with 200 µg/mL in the presence of S9 mix.

In this study, in the absence of S9 mix toxic effects indicated by clearly reduced cell numbers or mitotic indices were observed in all experimental parts. In detail, strongly reduced cell numbers were observed in experiment II after 18 hrs continuous treatment with 25 µg/mL (48 % of control). In all additional experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.

In contrary, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied test item concentration being in the range of test item precipitation.

In both cytogenetic experiments, in the absence and the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).

In both experiments, EMS (200 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, being in the range of the historical control data.

In conclusion, it can be stated that under the experimental conditions reported, the test item 2,2-Dimethyl-3-lauroyloxy-propanal did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations (without metabolic activation) and to clearly precipitating concentrations (with metabolic activation).

 

Cell gene mutation assay:

The test item 2,2-Dimethyl-3-Iauroyloxy-propanal (Aldehyde L) was tested for genetic toxicity in vitro in a cell gene mutation test with mouse lymphoma cells.

In this mutation assay the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2E05/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments.

In the Assay 1, in the absence and also in presence of S9 Mix dose-related toxicity of the test item was observed based on both, on the harmonised relative survival (harmonised RS) and on the relative total growth (RTG) values. There were differences between the harmonised RS and the RTG values in absence and in presence of S9 Mix. In absence of exogenous metabolic activation the concentration level of 50 μg/mL; in presence of S9 Mix the concentration levels of 140 and 120 μg/mL were out of the analysable range, but the guideline criteria for the number of analysable concentration levels (at least four) as well as the required cytotoxicity degree (10-20% relative survival or relative total growth) at the maximum concentration (in this case maximum analyzable concentration) were fulfilled.

At the concentration level of 120 μg/mL (+S9 Mix) the obtained mutation frequency differed statistically significantly from that of the vehicle control (Dunnett’s Test,α= 0.05), but remained far below the GEF criterion for positive call and at this concentration the high level of toxicity (~97 %, based on the harmonised RS) was observed, therefore the observed significance was not taken into consideration as biological relevant.

The phases of the Assay 2 accomplished the necessary concentration ranges for a valid assay. There were at least four analyzable concentration levels, where the highest concentration level was based on the required level of the caused cytotoxicity (80-90 %). The concentration levels examined were slightly modified in presence of S9 Mix, because of the observed high degree of toxicity obtained in the Assay 1. Despite the investigated slightly lower concentration levels the highest concentration level 115 μg/mL dropped out of the analyzable concentration range, due to the narrow test item effect concentration range.

In the Assay 2 in presence and also in absence of exogenous metabolic activation there were one or two concentration levels where the obtained mutation frequencies statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). The changes of the mutation frequencies were not dose-related, not biologically relevant, and the GEF criterion for positive call was not attained in any case.

Justification for classification or non-classification

Based on results obtained from in vitro genetic toxicity testing, Aldehyde L was not classified or labeled according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.